Structural basis for directional chitin biosynthesis.

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2-4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a 'gate lock' that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.

Characterization of Different Salt Forms of Chitooligosaccharides and Their Effects on Nitric Oxide Secretion by Macrophages.

In this paper, chitooligosaccharides in different salt forms, such as chitooligosaccharide lactate, citrate, adipate, etc., were prepared by the microwave method. They were characterized by SEM, FTIR, NMR, etc., and the nitric oxide (NO) expression was determined in RAW 264.7 cells. The results showed that pure chitooligosaccharide was an irregular spherical shape with rough surface, and its different salt type products are amorphous solid with different honeycomb sizes. In addition to the characteristic absorption peaks of chitooligosaccharides, in FTIR, the characteristic absorption of carboxyl group, methylene group, and aromatic group in corresponding acid appeared. The characteristic absorption peaks of carbon in carboxyl group, hydrogen and carbon in methyl, methylene group, and aromatic group in corresponding acid also appeared in NMR. Therefore, the sugar ring structure and linking mode of chitooligosaccharides did not change after salt formation of chitooligosaccharides. Different salt chitooligosaccharides are completely different in promoting NO secretion by macrophages, and pure chitooligosaccharides are the best.

Progress in chitin analytics.

Chitin is the second most abundant biopolymer and functions as the main structural component in a variety of living organisms. In nature, chitin rarely occurs in a pure form, but rather as nanoorganized chitin-proteins, chitin-pigments, or chitin-mineral composite biomaterials. Although chitin has a long history of scientific studies, it is still extensively investigated for practical applications in medicine, biotechnology, and biomimetics. The complexity of chitin has required the development of highly sensitive analytical methods for its identification. These methods are crucial for furthering disease diagnostics as well as advancing modern chitin-related technologies. Here we provide a summary of chitin identification by spectroscopic (NEXAFS, FTIR, Raman, NMR, colorimetry), chromatographic (TLC, GC, HPLC), electrophoretic (HPCE), and diffraction methods (XRD, WAXS, SAXS, HRTEM-SAED). Biochemical and immunochemical (ELISA, immunostaining) methods are described with respect to their medical application. This review outlines the history as well as the current progress in the analytical methods for chitin identification.

Abundant Chitinous Structures in Chilostomella (Foraminifera, Rhizaria) and Their Potential Functions.

Benthic foraminifera, members of Rhizaria, inhabit a broad range of marine environments and are particularly common in hypoxic sediments. The biology of benthic foraminifera is key to understanding benthic ecosystems and relevant biogeochemical cycles, especially in hypoxic environments. Chilostomella is a foraminiferal genus commonly found in hypoxic deep-sea sediments and has poorly understood ecological characteristics. For example, the carbon isotopic compositions of their lipids are substantially different from other co-occurring genera, probably reflecting unique features of its metabolism. Here, we investigated the cytoplasmic and ultrastructural features of Chilostomella ovoidea from bathyal sediments of Sagami Bay, Japan, based on serial semi-thin sections examined using an optical microscope followed by a three-dimensional reconstruction, combined with TEM observations of ultra-thin sections. Observations by TEM revealed the presence of abundant electron-dense structures dividing the cytoplasm. Based on histochemical staining, these structures are shown to be composed of chitin. Our 3D reconstruction revealed chitinous structures in the final seven chambers. These exhibited a plate-like morphology in the final chambers but became rolled up in earlier chambers (toward the proloculus). These chitinous, plate-like structures may function to partition the cytoplasm in a chamber to increase the surface/volume ratio and/or act as a reactive site for some metabolic functions.

Characteristics of chitin extracted from black soldier fly in different life stages.

Chitin was collected and extracted along different lifecycle stages of the Black Soldier Fly (BSF) (larvae, prepupae, pupae, flies, shedding & cocoons). The chitin content in the collected biomass ranged between 8% and 24%, with sheddings and cocoons being most rich in chitin. Purified chitin was subjected to a physicochemical evaluation based on FTIR, XRD, and TGA as well as a deacetylation step. The data indicated that BSF chitin was α-chitin with FTIR profiles matching closely to shrimp chitin and showing some differences compared to squid pen chitin (ß-chitin). Small physicochemical differences were observed among the different BSF samples. Prepupae and cocoon chitin was more crystalline while chitin from larvae and sheddings had a lower thermal degradation temperature. In addition, sheddings were more difficult to purify. Further processing to chitosan showed that a deacetylation degree of 89% could be obtained for all samples after 3 h, although sheddings were found to be less reactive in the deacetylation process. Overall, the small differences in physicochemical properties that were detected between the BSF chitin samples did not prevent further processing of chitin to chitosan with the same degree of deacetylation via the same treatment.

Potential of discarded sardine scales (Sardina pilchardus) as chitosan sources.

The random discharge of marine fish waste into the coast generates environmental pollution. However, a better valorization of these by-products leads to the extraction of sustainable biomolecules. Chitosan is a natural biopolymer that can be produced from various marine by-products, in particular the crustacean shells, crabs, and fish scales. The aim of this current study is the extraction of chitin and characterization of chitosan obtained after a deacetylation reaction from sardine scales (S. pilchardus) as a new marine source. The ß form of chitin extracted undergoes deacetylation in 40% NaOH at 121°C for 20 min. The chemical structure of obtained chitosan was characterized based on Fourier transforms infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), Scanning electron microscope (SEM), and Energy-dispersive X-ray spectroscopy (EDS). The physicochemical properties of obtained chitosan such as the ash, moisture, nitrogen, solubility, molecular weight, fat, and water-binding capacity were also determined. According to the results of FTIR and XRD analysis, the degree of deacetylation (DDA), and the crystalline index (CrI) value of obtained chitosan is respectively about 87% and 95%. The SEM and EDS analysis revealed respectively fibrillar and pleated morphology with the presence of three major elements characterizing the chitosan, which are C, O, and N. The physicochemical analysis showed that the rate of ash, moisture, and nitrogen in obtained chitosan were respectively about 0.10, 0.34, and 7%. The solubility, molecular weight, fat, and water-binding capacity of produced chitosan were found to be 93%, 5.86 kDa, 310, and 510% respectively. Sardina pilchardus scales could be considered a promising and alternative source of chitin and chitosan, which will be applicable in a large number of fields. Implications: Direct rejection of marine biowaste as fish scales in nature, port, or fish processing plants, is a dramatic problem that is growing day after day. These uncontrollable discharges cause marine pollution and promote bacterial growth, which leads to a degradation of the soil and air quality. Taking into account the objectives of sustainable development, better development of these by-products would make it possible to produce valuable biomaterials that will be applied in various fields and which have benefits for the environment and humans. The central objective of this research is accentuated on the enhancement of Sardina pilchardus scales; by the conversion of chitin into chitosan and the determination of its physicochemical characterization. The obtained chitosan from Sardina pilchardus scales could be applied in the agricultural and food industry.

Structural and functional analyses of organic molecules regulating biomineralization.

Biomineralization by living organisms are common phenomena observed everywhere. Molluskan shells are representative biominerals that have fine microstructures with controlled morphology, polymorph, and orientation of CaCO3 crystals. A few organic molecules involved in the biominerals play important roles in the formation of such microstructures. Analyses of structure-function relationships for matrix proteins in biominerals revealed that almost all matrix proteins have an acidic region for the binding of calcium ion in CaCO3 crystals and interaction domains for other organic molecules. On the other hand, biomineralization of metal nanoparticles by microorganisms were also investigated. Gold nanoparticles and quantum dots containing cadmium were successfully synthesized by bacteria or a fungus. The analyses of components revealed that glycolipids, oligosaccharides, and lactic acids have key roles to synthesize the gold nanoparticle in Lactobacillus casei as reductants and dispersants. These researches about biomineralization will give new insights for material and environmental sciences in the human society.

Surface modification of α-chitin using an acidic treatment followed by ultrasonication: Measurements of their sorption properties.

The biopolymer α-chitin is a promising raw source that can be used as a low-cost material for environmental applications. Nevertheless, its low surface properties and high crystallinity limit its use. Recent developments include surface modification as one of the most promising strategies for the application of α-chitin. To this end, we used an acidic treatment, followed by ultrasonication, to modify the α-chitin surface and improve its sorption characteristics to achieve the above goal. Structural analysis and measurement of the physicochemical properties (chemical structure and thermal degradation) of α-chitin, before and after surface modification, indicated no significant changes. However, specific surface area, morphology, surface charge, crystallinity and study of the sorption of methylene blue (MB) from aqueous solution demonstrated surface modification. It was established that the SBET of modified α-chitin increased to 110.7 m2/g and the crystallinity index decreased to 48%. Interestingly, the modified α-chitin could easily adsorb organic dye from an aqueous solution. The experimental adsorption capacity of the resulting α-chitin after surface modification reached the value of about 95 mg/g.

Guanine crystals regulated by chitin-based honeycomb frameworks for tunable structural colors of sapphirinid copepod, Sapphirina nigromaculata.

Sapphirinid copepods, which are marine zooplankton, exhibit tunable structural colors originating from a layered structure of guanine crystal plates. In the present study, the coloring portion of adult male of a sapphirinid copepod, Sapphirina nigromaculata, under the dorsal body surface was characterized to clarify the regulation and actuation mechanism of the layered guanine crystals for spectral control. The coloring portions are separated into small domains 70-100 µm wide consisting of an ordered array of stacked hexagonal plates ~1.5 µm wide and ~80 nm thick. We found the presence of chitin-based honeycomb frameworks that are composed of flat compartments regulating the guanine crystal plates. The structural color is deduced to be tuned from blue to achromatic via yellow and purple by changing the interplate distance according to vital observation and optical simulation using a photonic array model. The framework structures are essential for the organization and actuation of the particular photonic arrays for the exhibition of the tunable structural color.

Nested helicoids in biological microstructures.

Helicoidal formations often appear in natural microstructures such as bones and arthropods exoskeletons. Named Bouligands after their discoverer, these structures are angle-ply laminates that assemble from laminae of chitin or collagen fibers embedded in a proteinaceous matrix. High resolution electron microscope images of cross-sections through scorpion claws are presented here, uncovering structural features that are different than so-far assumed. These include in-plane twisting of laminae around their corners rather than through their centers, and a second orthogonal rotation angle which gradually tilts the laminae out-of-plane. The resulting Bouligand laminate unit (BLU) is highly warped, such that neighboring BLUs are intricately intertwined, tightly nested and mechanically interlocked. Using classical laminate analysis extended to laminae tilting, it is shown that tilting significantly enhances the laminate flexural stiffness and strength, and may improve toughness by diverting crack propagation. These observations may be extended to diverse biological species and potentially applied to synthetic structures.

Extraction, characterization and comparison of chitins from large bodied four Coleoptera and Orthoptera species.

Chitins were extracted from large insect species of order Coleoptera (Lucanus cervus (Linnaeus, 1758) (Lucanidae) and Polyphylla fullo (Linnaeus, 1758) (Scarabaeidae) and order Orthoptera (Bradyporus (Callimenus) sureyai Ünal, 2011) (Tettigonidae) and Gryllotalpa gryllotalpa (Linnaeus, 1758) (Gryllotalpidae)) for the first time. Fourier Transform Infrared Spectrometry (FT-IR) confirms that isolation of chitin is successful. Yields of chitins on dry basis from P. fullo, L. cervus, G. gryllotalpa and B. (C.) sureyai are 11.3%, 10.9%, 10.1% and 9.8% respectively. Thermogravimetric Analysis (TGA) showed a variety of thermal stability of chitin samples from 614 °C to 748 °C with a small percent of ash. X-ray diffraction (XRD) data showed a crystallinity index percent from 80.6% to 85.2%. Scanning Electron Microscope (SEM) was examined for surface characterization determining as fibrous and porous for all species and changes from nm scales to µm scales. Elemental analysis has been applied to determine the elemental composition of chitin and nitrogen percent was relatively low for all specimens than expected. It is detected that examined insects have α-chitin form from XRD and FT-IR data. If these species can be grown in the laboratory, adults of them could be accepted as promising alternative chitin sources without negative effects on biodiversity.

Different Molecular Interaction between Collagen and α- or ß-Chitin in Mechanically Improved Electrospun Composite.

Although collagens from vertebrates are mainly used in regenerative medicine, the most elusive issue in the collagen-based biomedical scaffolds is its insufficient mechanical strength. To solve this problem, electrospun collagen composites with chitins were prepared and molecular interactions which are the cause of the mechanical improvement in the composites were investigated by two-dimensional correlation spectroscopy (2DCOS). The electrospun collagen is composed of two kinds of polymorphs, α- and ß-chitin, showing different mechanical enhancement and molecular interactions due to different inherent configurations in the crystal structure, resulting in solvent and polymer susceptibility. The collagen/α-chitin has two distinctive phases in the composite, but ß-chitin composite has a relatively homogeneous phase. The ß-chitin composite showed better tensile strength with ~41% and ~14% higher strength compared to collagen and α-chitin composites, respectively, due to a favorable secondary interaction, i.e., inter- rather than intra-molecular hydrogen bonds. The revealed molecular interaction indicates that ß-chitin prefers to form inter-molecular hydrogen bonds with collagen by rearranging their uncrumpled crystalline regions, unlike α-chitin.

Cricket tympanal organ revisited: morphology, development and possible functions of the adult-specific chitin core beneath the anterior tympanal membrane.

Vertebrates and insects are phylogenetically separated by millions of years but have commonly developed tympanal membranes for efficiently converting airborne sound to mechanical oscillation in hearing. The tympanal organ of the field cricket Gryllus bimaculatus, spanning 200 µm, is one of the smallest auditory organs among animals. It indirectly links to two tympana in the prothoracic tibia via tracheal vesicles. The anterior tympanal membrane is smaller and thicker than the posterior tympanal membrane and it is thought to have minor function as a sound receiver. Using differential labeling of sensory neurons/surrounding structures and three-dimensional reconstructions, we revealed that a shell-shaped chitin mass and associated tissues are hidden behind the anterior tympanal membrane. The mass, termed the epithelial core, is progressively enlarged by discharge of cylindrical chitin from epithelial cells that start to aggregate immediately after the final molt and it reaches a plateau in size after 6 days. The core, bridging between the anterior tracheal vesicle and the fluid-filled chamber containing sensory neurons, is supported by a taut membrane, suggesting the possibility that anterior displacements of the anterior tracheal vesicle are converted into fluid motion via a lever action of the core. The epithelial core did not exist in tympanal organ homologs of meso- and metathoracic legs or of nymphal legs. Taken together, the findings suggest that the epithelial core, a potential functional homolog to mammalian ossicles, underlies fine sound frequency discrimination required for adult-specific sound communications.

New Source of 3D Chitin Scaffolds: The Red Sea Demosponge Pseudoceratina arabica (Pseudoceratinidae, Verongiida).

The bioactive bromotyrosine-derived alkaloids and unique morphologically-defined fibrous skeleton of chitin origin have been found recently in marine demosponges of the order Verongiida. The sophisticated three-dimensional (3D) structure of skeletal chitinous scaffolds supported their use in biomedicine, tissue engineering as well as in diverse modern technologies. The goal of this study was the screening of new species of the order Verongiida to find another renewable source of naturally prefabricated 3D chitinous scaffolds. Special attention was paid to demosponge species, which could be farmed on large scale using marine aquaculture methods. In this study, the demosponge Pseudoceratina arabica collected in the coastal waters of the Egyptian Red Sea was examined as a potential source of chitin for the first time. Various bioanalytical tools including scanning electron microscopy (SEM), fluorescence microscopy, FTIR analysis, Calcofluor white staining, electrospray ionization mass spectrometry (ESI-MS), as well as a chitinase digestion assay were successfully used to confirm the discovery of α-chitin within the skeleton of P. arabica. The current finding should make an important contribution to the field of application of this verongiid sponge as a novel renewable source of biologically-active metabolites and chitin, which are important for development of the blue biotechnology especially in marine oriented biomedicine.

Biomacromolecules within bivalve shells: Is chitin abundant?

Bivalve shells are inorganic-organic nanocomposites whose material properties outperform their purely inorganic mineral counterparts. Most typically the inorganic phase is a polymorph of CaCO3, while the organic phase contains biopolymers which have been presumed to be chitin and/or proteins. Identifying the biopolymer phase is therefore a crucial step in improving our understanding of design principles relevant to biominerals. In this work we study seven shells; four are examples of nacroprismatic shells (Alathyria jacksoni, Pinctada maxima, Hyriopsis cumingii and Cucumerunio novaehollandiae), one homogeneous (Arctica islandica), and two are crossed lamellar (Callista kingii, Tridacna gigas). Both intact shells, their organic extracts as isolated after decalcification in acid, and the periostracum overlay have been studied by solid-state CP-MAS NMR, FTIR, SEM and chemical analysis. In none of the shells examined in this work do we find a significant contribution to the organic fraction from chitin or its derivatives despite popular models of bivalve biomineralization which assume abundant chitin in the organic fraction of mollusk bivalve shells. In each of the nacroprismatic extracts the 13C NMR spectra represent similar proteinaceous material, Ala and Gly-rich and primarily organized as ß-sheets. A different, yet highly conserved protein was found in the periostracum covering each of the three nacreous shells studied. The Arctica islandica shells with homogeneous microstructure contained proteins which do not appear to be silk-like, while in the crossed lamellar shells we extracted too little organic matter to characterize. STATEMENT OF SIGNIFICANCE: Hydrophobic macromolecules are structural components within the calcareous inorganic matrix of bivalve shells and are responsible for enhanced materials properties of the biominerals. Prevalent models suggest that chitin is such major hydrophobic component. Contrary to that we show that chitin is rare within the hydrophobic biopolymers which primarily consist of proteinaceous matter with structural motifs as silk-like ß-sheets, or others yet to be determined. Recognizing that diverse proteinaceous motifs, devoid of abundant chitin, can yield the optimized mechanical properties of bivalve shells is critical both to understand the mechanistic pathways by which they regulate biomineralization and for the design of novel bioinspired materials.

The demosponge Pseudoceratina purpurea as a new source of fibrous chitin.

Among marine demosponges (Porifera: Demospongiae), only representatives of the order Verongiida have been recognized to synthetize both biologically active substances as well as scaffolds-like fibrous skeletons made of structural aminopolysaccharide chitin. The unique 3D architecture of such scaffolds open perspectives for their applications in waste treatment, biomimetics and tissue engineering. Here, we focus special attention to the demosponge Pseudoceratina purpurea collected in the coastal waters of Singapore. For the first time the detailed description of the isolation of chitin from the skeleton of this sponge and its identification using diverse bioanalytical tools were carried out. Calcofluor white staining, FTIR analysis, electrospray ionization mass spectrometry (ESI-MS), SEM, and fluorescence microscopy as well as a chitinase digestion assay were applied in order to confirm with strong evidence the finding of alpha-chitin in the skeleton of P. purpurea. We suggest that the discovery of chitin within representatives of Pseudoceratinidae family is a perspective step in evaluation of these verongiid sponges as novel renewable sources for both chitin and biologically active metabolites, which are of prospective use for marine oriented biomedicine and pharmacology, respectively.

An inclusive physicochemical comparison of natural and synthetic chitin films.

Natural and synthetic chitin films, obtained from the same source were produced and their physicochemical properties were examined comparatively. Firstly, natural chitin film was obtained from elytra of an insect (Oryctes nasicornis L.) and purity of the obtained chitin film (degree of acetylation: 79±2%) was demonstrated by solid state 13C nuclear magnetic resonance (13C NMR). Then, the synthetic film was produced by dissolving of natural chitin film in LiCl-DMAc. The obtained natural and synthetic films were characterized by AFM, TGA, DSC, FTIR, mechanical properties, light transmission and contact angle. The analyses result demonstrated that natural chitin film lost very important properties such as high thermal stability, transparency, nanofibrous nature, tensile strength, Young's modulus and hydrophobicity after transforming the synthetic film.

Crystal structure and thermodynamic dissection of chitin oligosaccharide binding to the LysM module of chitinase-A from Pteris ryukyuensis.

We determined the crystal structure of a LysM module from Pteris ryukyuensis chitinase-A (PrLysM2) at a resolution of 1.8 Å. Structural and binding analysis of PrLysM2 indicated that this module recognizes chitin oligosaccharides in a shallow groove comprised of five sugar-binding subsites on one side of the molecule. The free energy changes (ΔGr°) for binding of (GlcNAc)6, (GlcNAc)5, and (GlcNAc)4 to PrLysM2 were determined to be -5.4, -5,4 and -4.6 kcal mol-1, respectively, by ITC. Thermodynamic dissection of the binding energetics of (GlcNAc)6 revealed that the driving force is the enthalpy change (ΔHr° = -11.7 ± 0.2 kcal/mol) and the solvation entropy change (-TΔSsolv° = -5.9 ± 0.6 kcal/mol). This is the first description of thermodynamic signatures of a chitin oligosaccharide binding to a LysM module.

Structural and optical investigation on the wings of Idea malabarica (Moore, 1877).

The nanostructures on the wings of Idea malabarica (Moore, 1877) were analysed using scanning electron microscopy, energy dispersive X-ray spectroscopy, atomic force microscopy, Fourier transform-infrared spectroscopy, and reflectance measurements. The chemical and morphological analyses revealed the chitin-based intricate nanostructures. The influence of the nanostructures on the wetting characteristics of the wing was investigated using optical imaging. Applying the Maxwell-Garnet approximation to the porosities within the nanostructures, the refractive indices, which relate the reflectance response, were estimated. It was concluded that the colour seen on the wings of the Idea malabarica originate from the nanostructural configurations of the chitin-based structures and the embedded pigment.

Crystal defects induced by chitin and chitinolytic enzymes in the prismatic layer of Pinctada fucata.

Biomineralization, in which organisms create biogenic hard tissues, with hardness or flexibility enhanced by organic-inorganic interaction is an interesting and attractive focus for application of biomimetic functional materials. Calcites in the prismatic layer of Pinctada fucata are tougher than abiotic calcites due to small crystal defects. However, the molecular mechanism of the defect formation remains unclear. Here, chitin and two chitinolytic enzymes, chitinase and chitobiase, were identified as organic matrices related to for the formation of small crystal defects in the prismatic layer. Experiments with a chitinase inhibitor in vivo showed chitinase is necessary to form the prismatic layer. Analysis of calcite crystals, which were synthesized in a chitin hydrogel treated with chitinolytic enzymes, by electron microscopy and X-ray diffraction showed that crystal defects became larger as chitin was more degraded. These results suggest that interactions between chitin and calcium carbonate increase as chitin is thinner.